Technique for the Kinetic Characterization of the Metabolic Reactions of Hepatocytes in Adhesion Culture

Abstract

In this paper, we report on the development of a technique for the kinetic characterization of the metabolic reactions of liver cells in adhesion culture. The technique is based on the use of a continuous-flow bioreactor which is designed and operated in such a way as to ensure a uniform distribution of metabolite at the cell site: hence, the metabolite concentration at the surface of cells cultured in adhesion at the bottom of the bioreactor equals that in the stream leaving the bioreactor. Under steady conditions, the rate of a given cell reaction is directly estimated from the metabolite concentration difference in the streams entering and leaving the bioreactor and can be correctly related to the actual concentration at the cell surface. Such a technique was used for a preliminary investigation of the kinetics of ammonia elimination, urea synthesis, and phenolsulfonphthalein (PSP) elimination by primary rat hepatocytes cultured in adhesion on collagen, with respect to ammonia and PSP concentration, respectively. The rate at which the hepatocytes eliminated ammonia increased with increasing ammonia concentrations according to a Michaelis-Menten kinetics. The hepatocytes synthesized urea also in the absence of ammonia in the medium: as ammonia concentration increased, the cells synthesized urea at a rate that increased according to a saturation kinetics. In the concentration range investigated, the hepatocytes eliminated PSP at a rate that increased linearly with the actual PSP concentration in the medium. Such kinetic information can be coupled to the mechanism of metabolite transport in a hybrid liver support device to yield an effective device design for the treatment of acute liver failure.

Flexibility of the hepatic zonation of carbon and nitrogen fluxes linked to lactate and pyruvate transformations in the presence of ammonia

“…3) or pyruvate and ammonia (50 min perfusion time in Fig. In hepatocytes, ammonia uptake and urea production have also been found to obey apparent Michaelis-Menten kinetics with respect to the extracellular ammonia concentrations (10). 3 and 4).…”

Perfusion culture of hepatocytes within galactose‐derivatized biodegradable poly(lactide‐co‐glycolide) scaffolds prepared by gas foaming of effervescent salts

“…Previous studies reported that oxygen uptake rate of primary hepatocytes was critical in the early stage of cell attachment process. 35,36 Various continuous flow and perfusion culture systems were utilized to overcome the mass transfer limitations occurred in the cell/scaffold constructs. [37][38][39] The perfusion culture system, facilitating supply of nutrients and oxygen to the seeded cells and removal of metabolic products and degradation products from biodegradable scaffolds, showed improved cell viability and functions for various tissue regenerations.…”

Three-dimensional Numerical Modeling and Computational Fluid Dynamics Simulations to Analyze and Improve Oxygen Availability in the AMC Bioartificial Liver

“…For this reason, a lower O 2 consumption may be expected as the specific O 2 uptake rate per cell is reported to decrease at increasing cell densities 30. On the other hand, O 2 consumption may rise significantly when a metabolic load is applied to the cells 4. As such, the proposed consumption characteristics are justified by assuming these values to represent the most limiting case for oxygen availability.…”